Combination therapies for treatment of cancer

ABSTRACT

A method for treatment of prostate cancer or benign prostate hyperplasia by combination therapy comprising administering to a patient an androgen-deprivation therapy agent and a bacteriochlorophyll derivative followed by photodynamic therapy (PDT) or vascular-targeted photodynamic therapy (VTP).

RELATED APPLICATION

This application claims priority to and the benefit of provisionalapplication U.S. 62/472,126 filed Mar. 16, 2017, which is incorporatedby reference herein in its entirety as if fully disclosed therein.

FIELD OF THE INVENTION

The present invention relates to treatment of cancer and to combinationtherapies therefor. In particular, the invention relates to treatment ofprostate cancer or benign prostate hyperplasia by combination therapy.

Definitions and Abbreviations

ADT: androgen deprivation therapy; AR: androgen receptor; Bchl:bacteriochlorophyll: Bchl-D: bacteriochlorophyll derivative; BPH: benignprostate hyperplasia; GSEA: Gene Set Enrichment Analysis; LNCaP-AR: aprostate cancer cell line that overexpresses the androgen receptor;Padeliporfin: generic name for TOOKAD; PCa: Prostate cancer; PDT:photodynamic therapy; PSA: prostate-specific antigen; ROS: reactiveoxygen species; TOOKAD: brand name for WST11 or padeliporfindi-potassium; TUNEL: terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling; VCaP: a cell-based model systemof human prostate cancer; VTP: vascular targeted photodynamic therapy;WST11: the water-soluble Bchl-D Palladium3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13¹-(2-sulfoethyl)amide dipotassium salt.

BACKGROUND

Prostate cancer represents the most common cause of cancer-relateddeaths in men, particularly of age 50 or more. Prostate cancer usuallydevelops slowly, so signs may not be seen for many years. The symptomsoften only become apparent when the prostate is large enough to affectthe urethra, but these symptoms can also be caused by benign prostatichyperplasia (BPH), a non-cancerous growth of the prostate.

Survival for men with prostate cancer directly depends on the stage andgrade of the disease at the time of diagnosis, which is performed bymeasuring the level of serum prostate-specific antigen (PSA) andconducting a digital rectal examination. The risk of prostate cancer iscategorized as low, intermediate and high, based on clinical stage,serum PSA level and Gleason score.

After prostate cancer diagnosis, staging provides important informationabout the extent of cancer in the body. This is done by measuring theextent of the primary tumor, checking Whether the cancer has spread tonearby lymph nodes and/or metastasized to other parts of the body,determining the PSA level at the time of diagnosis and determining theGrade group (based on the Gleason score), which is a measure of howlikely the cancer is to grow and spread quickly, as determined by theresults of the prostate biopsy (or surgery).

Based on the diagnosis and staging determination, an anticipatedresponse to treatment may be reached. Current treatment choices forprostate cancer include watchful waiting or active surveillance,surgery, radiation therapy, cryotherapy, hormone therapy, chemotherapy,vaccine treatment and bone-directed treatment.

For small, slow-growing cancers localized just in the prostate, watchfulwaiting or active surveillance without specific therapy is an option,particularly at the very low grade and risk. Only when during activesurveillance it is noted that the cancer progressed to intermediate(grade 2) or high (grade 3) risk (for example, involving >3 Gleasonscore foci) and therefore is a more serious form of cancer, or thecancer is likely to have spread outside the prostate (based on magneticresonance imaging (MRI) and MRI targeted biopsy)), active treatment isstarted by radical therapies such as surgery, radiation therapy,cryotherapy, hormone therapy, or chemotherapy.

Hormone therapy, also called androgen deprivation therapy (ADT) orandrogen suppression therapy, has the goal to reduce levels of malehormones, called androgens, in the body, or to stop them fromstimulating prostate cancer cells to grow. The main androgens in thebody are testosterone and dihydrotestosterone (DHT). Most of theandrogens are made by the testicles, but the adrenal glands also make asmall amount, Lowering androgen levels or stopping them from gettinginto prostate cancer cells often makes prostate cancers shrink or growmore slowly for a time, but hormone therapy alone does not cure prostatecancer.

Radical therapies of prostate cancer may be unnecessarily aggressive andare associated with important side effects and a risk of overtreatment.Therefore, there has been interest in developing focal therapies thatare less aggressive than radical therapies as an alternative treatmentoption for patients with low-risk prostate cancer [Cathelineaux andSanchas-Salas, 2016].

Photodynamic therapy (PDT) in general and vascular-targeted PDT (VTP) inparticular using novel bacteriochlorophyll derivatives were shown toselectively ablate localized solid tumors in different targets (WO00/33833; WO 2004/045492) and are suitable for minimally-invasiveablative treatments for prostate cancer.

Vascular targeted photodynamic therapy (VTP) using padeliporfin as aphotosensitizer (TOOKAD® Soluble, WST11, padeliporfin) in associationwith a low power near-infrared laser light in the presence of oxygendestroys targeted tissues. The photosensitizer absorbs light andtransfers energy to oxygen molecules creating reactive oxygen species(ROS) such as super oxide and hydroxyl radicals. As WST11 is retained inthe tumor vasculature until clearance, generation of intravascular ROSinitially induces local vascular destruction resulting in tumor cellnecrosis. Padeliporfin is intravenously infused and circulatessystemically while only the cancerous lobe of the prostate isilluminated by trans-perineal optic fibers. To treat localized prostatecancer, these probes can be positioned to deliver PDT or VTP to either aportion of, or to the entire, prostate gland. VTP induces irreversibledamage to endothelial cells which is quickly followed by a cascade ofevents including thrombosis, blood stasis and vessel occlusion, leadingto tumor necrosis [Ashur et al., 2009; Brandis et al., 2005; Borle etal., 2003].

Positive outcomes from patients with low-risk, localized PCa (Gleasonpattern 3 with no previous treatment) treated with WST11NTP haverecently been reported in European multi-center phase 2 and 3 studies.In follow up biopsies at 6 months after prostate hemiablation, 80.6% ofthe patients were negative for cancer [Azzouzi et al., 2015] and therewas a decreased disease progression at 24 months when compared to activesurveillance (28% versus 58% respectively, HR 0.34, 95% CI 0.24-0.46;p<0.0001) [Azzouzi et al., 2017]. After a median follow-up of 68 months,82% of patients treated with WST11NTP were free of clinicallysignificant cancer in the treated lobes and 76% of the treated patientshad avoided a need for subsequent radical therapy [Lebdai et al., 2017].Thus, after successful clinical trials (Azzouzi et al., 2015, 2017),TOOKAD/padeliporfin/WST11 has been recently approved for use in earlystage prostate cancer treatment.

It would be very desirable to improve the efficacy of VTP with acomplementary, targeted combination therapy. Furthermore, combinationtherapy may allow for the extension of VTP treatment to additionalcohorts of patients with intermediate or high-risk localized prostatecancer.

SUMMARY OF THE INVENTION

The present invention relates to a method for treatment of prostatecancer or benign prostate hyperplasia by combination therapy comprisingadministering to a patient in need thereof: (i) a therapeuticallyeffective amount of an androgen-deprivation therapy (ADT) agent(hereinafter “ADT agent”); and (ii) a therapeutically effective amountof a bacteriochlorophyll derivative (herein after “Bchl-D”) followed byphotodynamic therapy (PDT) or vascular-targeted photodynamic therapy(VTP).

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the United States Patent andTrademark Office upon request and payment of the necessary fee.

FIGS. 1A-1B depict the transcriptome analysis of LNCaP-AR human prostatecancer xenografts post-VTP treatment with WST11. FIG. 1A: Top rankedGSEA pathways among the gene sets HALLMARK (left) and Canonical Pathways(C2) (right). FIG. 1B: Enrichment plots from the GSEA with normalizedenrichment scores (NES) for androgen response pathways within both genesets at 6 hr post-VTP (n=4) vs. control (n=4).

FIG. 2 shows that ADT in combination with WST11/VTP displays potentialenhanced effects: LNCaP-AR human prostate cancer model in nude mice.Panel A: Treatment scheme showing administration of a sole dose ofdegarelix (0.5-1.0 mg per mouse) 3 days prior to VTP, and ofenzalutamide (30 mg/kg) daily for two weeks total both before and afterVTP. Panel B: Mice bearing LNCaP-AR tumors were randomly assigned to 6cohorts: control (n=7), degarelix (n=9), enzalutamide (n=10), VTP (n=8),degarelix+VTP (n=9), and enzalutamide+VTP (n=9) and tumor size wasmeasured weekly.

FIGS. 3A-3B show the efficacy of the ADT and WST11/VTP combination inthe LNCaP-AR human prostate cancer model in nude and SCID mice. FIG. 3A:Degarelix and WST11 VTP combination on tumor growth in athymic nudemice. Mice bearing LNCaP-AR tumors were randomly assigned to 4 cohorts:control (n=7), degarelix (n=9), VTP (n=8), and degarelix+VTP (n=9) andtumor size was measured weekly. The combination treatment suppressedtumor growth more efficiently (p<0.01 for combination vs degarelix,p<0.005 for combination vs VTP). FIG. 3B: Combination of degarelix andVTP on tumor growth in SCID mice. Mice bearing LNCaP-AR tumors wererandomly assigned to 4 cohorts: control (n=14), degarelix (n=14), VTP(n=17) and degarelix+VTP (n=16) and tumor size was measured weekly(p<0.0001, combination vs degarelix or VTP). Results were combined fromtwo separate experiments. One dose of degarelix was given at 3 daysprior to VTP.

FIG. 4 shows the efficacy of ADT and WST11/VTP combination in the VCaPhuman prostate cancer model in SCID mice. SCID mice bearing VCaP tumorswere randomly assigned to 4 cohorts: control (n=7), degarelix (n=9), VTP(n=8), degarelix+VTP (n=9) and tumor size was measured weekly. One doseof degarelix was given 3 days prior to VTP treatment. Combinationtherapy led to superior local tumor control compared to monotherapy(p<0.0001 for degarelix, p<0.005 for VTP).

FIGS. 5A-5C show superior local tumor control by ADT and WST11/VTPcombination correlated with a decline in serum PSA and intense TUNELstaining, but not in Ki67 staining. FIG. 5A: Fold changes of serum tPSAwere shown at days 1, 3, and 7 post-VTP. Decrease in PSA level wasstatistically significant in the mice treated with degarelix/VTP on allthree days (*p<0.05, ANOVA one way, non-parametric). FIG. 5B:Histological assessment of degarelix and VTP effects on day 7 post-VTPby H&E, AR and TUNEL. Magnifications of 4× and 20× are shown. FIG. 5C:ADT reduces tumor proliferation measured by Ki67. Histologicalassessment of degarelix effects on day 7 post-VTP by hematoxylin/eosinand Ki67. The number of Ki67 positive cells were reduced in degarelixtreated tumors compared to control tumors (p<0.05). Magnifications of 4×and 20× are shown. VTP and combination groups did not retain sufficientviable tumor tissue to quantitatively assess Ki67 staining.

FIGS. 6A-6B show that ADT reduces the number of vessels in tumors. FIG.6A: A representative H&E and CD31 IHC staining in control anddegarelix-treated tumors, with image analysis showing positive pixels inred (lower panels). FIG. 6B: The quantification of CD31 positive areasin the control group (n=12) and the degarelix-treated group (n=11) areshown. Degarelix treatment resulted in a 38% decrease in CD31 stainingarea compared to controls (P<0.05). Image analysis was performed usingQuPath software.

DETAILED DESCRIPTION OF THE INVENTION

While looking for potential druggable pathways active in prostate cancertumors exposed to VTP, the inventors identified by transcriptomeanalysis a compensatory, acute upregulation of AR pathway activationfollowing VTP treatment, indicating that inhibition of AR activity byandrogen deprivation therapy (ADT) could enhance the efficacy of VTPtreatment in prostate cancer.

In certain embodiments, the present invention thus relates to a methodfor treatment of prostate cancer or benign prostate hyperplasia bycombination therapy comprising administering to a patient in needthereof: (i) a therapeutically effective amount of anandrogen-deprivation therapy (ADT) agent (hereinafter “ADT agent”); and(ii) a therapeutically effective amount of a bacteriochlorophyllderivative (Bchl-D) followed by photodynamic therapy (PDT) orvascular-targeted photodynamic therapy (VTP) (hereinafter “Bchl-D VTP”).

In certain embodiments, the combination therapy provides an enhancedtherapeutic effect in the patient compared to the effect of the ADTagent or the Bchl-D followed by PDT or VTP, when each is administeredalone.

In certain embodiments, the combination therapy of the ADT agent and theBchl-D has a synergistic therapeutic effect.

In certain embodiments of the combination therapy, the ADT agent and theBchl-D agent are administered sequentially.

The ADT agent for use according to the method of the invention may be achemical castration agent or an antiandrogen agent.

In certain embodiments, the ADT agent is a chemical castration agent.These agents are luteinizing hormone-releasing hormone (LHRH, alsocalled gonadotropin-releasing hormone or GnRH) agonists or antagonistsand include drugs such as leuprolide, goserelin, triptorelin, histrelin,and degarelix.

In certain embodiments, the ADT agent is degarelix, a long-acting,gonadotropin-releasing hormone antagonist that results in a rapid onsetof medical castration [Broqua et al., 2002; Klotz et al., 2008]. It isuseful for treatment of castration-sensitive prostate cancer, includingcastration-sensitive metastatic prostate cancer.

In certain embodiments, the ADT agent is an antiandrogen agent. Theseagents include androgen antagonists or testosterone blockers. In certainembodiments, the antiandrogen agent is an androgen receptor (AR)antagonist that binds directly to, and blocks, the AR.

The AR antagonist may be a steroidal antiandrogen drug like cyproteroneacetate, megestrol acetate, chlormadinone acetate, medroxyprogesteroneacetate, spironolactone, and oxendolone, or a non-steroidal antiandrogendrug like flutamide, bicalutamide, nilutamide, topilutamide,enzalutamide, apalutamide and darolutamide.

In certain embodiments, the ADT agent is enzalutamide, a potentsecond-generation androgen receptor antagonist approved for thetreatment of men with castrate-resistant prostate cancer (CRPC).

Any Bchl-D shown to cause ablation of tumors may be used according tothe invention.

In certain embodiments, the Bchl-D for use according to the presentinvention is a water-soluble anionic bacteriochlorophyll derivative suchas those disclosed in the patent publications WO 2004/045492, U.S. Pat.Nos. 7,947,672, and 8,461,142, each and all of which are incorporated byreference herein in its entirety as if fully disclosed therein.

In certain embodiments, the Bchl-D for use according to the presentinvention is a water-soluble anionic bacteriochlorophyll derivativeconjugated with an RGD-containing peptide or RGD-peptidomimetic residuesuch as those disclosed in the patent publications WO 2008/023378, US2,012/0294801, WO 2009/107139, U.S. Pat. No. 8,815,213, WO 2010/046900and U.S. Pat. No. 8,673,270, each and all of which are incorporated byreference herein in its entirety as if fully disclosed therein.

In certain embodiments, the Bchl-D is an anionic Bchl-D of the formulaI:

wherein

-   -   M represents 2H or Pd;    -   R₁ is O—R₄ or —NHR₅, wherein R₄ is selected from the group        consisting of H, H⁺, an ammonium group or a monovalent metal        cation, and R₅ is an RGD-containing peptide or RGD        peptidomimetic residue;    -   R₂ is —O—C₁-C₆ alkyl;    -   R₃ is —NH—(CH₂)_(n)—SO₃ ⁻R₆ ⁺, wherein n is 2 or 3, and R₆ ⁺ is        a monovalent metal cation and    -   pharmaceutically acceptable salts and optical isomers thereof.

In certain embodiments, the monovalent metal cation represented by R₄ orby R₆₊ is Na⁺ or K⁺. In certain embodiments, R₂ is methoxy.

In certain embodiments, the anionic Bchl-D for use in the invention hasthe formula I wherein R₁ at position 17³ is OR₄, namely, the Bchl-D isnot conjugated to an RGD-containing peptide or RGD peptidomimeticresidue (“non-conjugated Bchl-D”). In certain embodiments, the Bchl-Dhas the formula I wherein R₁ is O—R₄, R₂ is methoxy, R₃ is—NH—(CH₂)_(n)—SO³⁻R₆₊, wherein n is 2, R₄ and R₆₊ are K⁺, and M is Pd,as represented by the compound Palladium3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13¹-(2-sulfoethyl)amide dipotassium salt (herein designated WST11). In certainembodiments, the Bchl-D has the formula I wherein R₁ is O—R₄, R₂ ismethoxy, R₃ is —NH—(CH₂)_(n)—SO³⁻R₆₊, wherein n is 2, R₄ and R⁶⁻ are K⁺,and M is 2H, as represented by the compound3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13¹-(2-sulfoethyl)amide dipotassium salt (herein designated STL-7012). Both Bchl-Ds aredisclosed in WO 2004/045492, in which the main inventor is Prof. AvigdorScherz, a co-inventor in the present application.

In certain embodiments, the non-conjugated Bchl-D, e.g., WST11, isintravenously infused to the patient and circulates systemically with noextravasation out of the circulation, namely, it is retained in thetumor vasculature, until clearance. Illumination confined to thecancerous lobe of the prostate using transperineal optic fibers inducesultrafast electron transfer to oxygen molecules in the circulation. Theresulting short lived super oxide and hydroxyl radicals initiate rapiddestruction of the targeted vasculature followed by a cascade ofbiological events that end with coagulative necrosis of the tumor. Thisis vascular-targeted PDT (VTP), a local ablation approach relying uponrapid, free radical-mediated destruction of tumor vasculature. For thisreason, in case of VTP, the area to be treated has to be locallyilluminated immediately, or at a time of up to 30 min, after theadministration of the non-conjugated Bchl-D.

Other non-conjugated anionic Bchl-Ds that can be used according to theinvention include the following compounds disclosed in WO 2004/045492:Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹-(3-sulfopropyl) amide dipotassium salt;3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13¹-(2-sulfoethyl)amide dipotassium salt;3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13¹-(3-sulfopropyl)amide dipotassium salt; and Palladium3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13¹-(2-sulfoethyl)amide potassium salt.

In certain embodiments, the Bchl-D for use in the invention is a Bchl-Dformula I conjugated to an RGD-containing peptide or RGD peptidomimeticresidue (hereinafter “conjugated Bchl-D”). These conjugated Bchl-Ds havethe formula I wherein R₁ at position 17³ is NH—R₅, wherein R₅ is anon-cyclic or cyclic RGD-containing peptide or RGD peptidomimeticresidue.

In certain preferred embodiments, the conjugated Bchl-D for use in theinvention is conjugated to a cyclic RGD-containing peptide or to acyclic RGD peptidomimetic residue. Examples of Bchl-Ds conjugated withcyclic RGD-containing peptide or cyclic RGD peptidomimetic residues foruse in the present invention include, but are not limited to, thosedisclosed in the publications WO 2008/023378 and WO 2010/046900.

In certain embodiments, the conjugated Bch-D is the3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin13¹-(2-sulfoethyl)amide-17³-(cycloRGDfK) amide potassium salt (hereindesignated STL-6014), wherein f indicates D-Phe, disclosed in WO2008/023378.

Other conjugated Bchl-Ds disclosed in WO 2008/023378 that can be usedaccording to the invention include, but are not limited to:

Palladium 3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin13¹42-sulfoethyl)amide-17³-(cycloRGDfK) amide potassium salt

Palladium 3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin13¹42-sulfoethyl)amide-17³-(cycloRADfK) amide potassium salt

Palladium 3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin13¹-(2-sulfoethyl)amide-17³-(cycloRGDf-N(Me)K)amide potassium salt Whena conjugated Bchl-D, e.g., STL-6014, is administered to the patient, itgoes to, and accumulates in, the tumor tissue. Then, upon localillumination, photodynamic generation of ROS is initiated byilluminating the tumor volume and close vicinity once the conjugatedBchl-D accumulates at sufficiently high concentrations and cleared fromthe surrounding tissue. This is tissue-targeted PDT and an area of thelocal for treatment is illuminated after some time, to allowaccumulation and optimal concentration of the conjugated Bchl-D in thetargeted tissue. This time may be of at least 4 h, preferably 6 h, afterthe administration of the conjugated Bchl-D is completed.

In certain embodiments, the patient's prostate cancer is a low riskprostate cancer. In certain embodiments, the patient's prostate canceris an intermediate risk prostate cancer. In certain other embodiments,the patient's prostate cancer is a high risk prostate cancer. In certainother embodiments, the patient's prostate cancer iscastration-sensitive.

The ADT agent and the Bchl-D may be administered sequentially accordingto several different regimens. In general, in a session of treatment forablation of a primary tumor, the PDT or VTP treatment comprises a soleadministration of the Bchl-D followed by illumination of an area of thelocal to be treated, and the ADT treatment comprises severaladministrations of the ADT agent at various determined time intervals.If necessary, the session may be repeated one or more times as needed.

In accordance with one regimen scheme, the ADT agent is administeredonce before the sole administration of the Bchl-D followed by PDT or VTPtreatment.

In certain embodiments, the present invention provides a method oftreating prostate cancer, said method comprising administering to apatient in need thereof: (i) a therapeutically effective amount of anADT agent; and (ii) a therapeutically effective amount of abacteriochlorophyll derivative (Bchl-D) followed by PDT or VTP, toprovide a combination therapy having an enhanced therapeutic effectcompared to the effect of the ADT agent or the Bchl-D PDT or VTP, eachadministered alone.

The terms “treating” and “treatment” or the phrase “to treat” as usedherein refers to any type of treatment that imparts a benefit to apatient afflicted with prostate cancer or benign prostate hyperplasia,including improvement in the condition of the patient (e.g., in one ormore symptoms), delay in the progression of the condition, prolongationof survival time, etc.

The term “therapeutically effective amount” as used herein for the ADTagent refers to an amount that is therapeutically effective in thetreatment of cancer as defined hereinabove when used in the combinationtherapy and administered as a single dose or in repeated doses accordingto the invention. The term “therapeutically effective amount” as usedherein for the Bchl-D refers to its capability of causing ablation ofthe tumor after performance of the PDT or VTP.

The studies described herein and in the figures show that the suggestedcombination of the two treatment modalities synergizes their impact,leading to primary tumor ablation. These studies and the evolvedprotocols can be translated into the clinical arena in a straightforwardmanner.

The invention will now be illustrated by the following non-limitativeExamples.

EXAMPLES Material and Methods

(i) Materials: Lyophilized WST11 was obtained from Steba Biotech (Cedex,France). A human prostate cancer cell line VCaP was purchased from ATCC(Manassas, Va.) and LNCaP-AR was kindly provided by Dr. Charles Sawyers(MSKCC). Both cell lines were tested negative for mycoplasma using theMycoAlert™ PLUS Assay from Lonza (Basel, Switzerland). LNCaP-AR cellswere cultured in RPMI supplemented with 10% FBS, 2 mmol/L L-glutaminewhile VCaP cells were cultured in DMEM with high glucose, 10% FBS and 2mmol/L L-glutamine. All the components for cell culture were from LifeTechnologies (Grand Island, N.Y.). Degarelix was purchased from FerringPharmaceuticals Inc. (Parsippany, N.J.).

(ii) Animal models: All animal work was performed in accordance with aprotocol approved by the Institutional Animal Care and Use Committee(IACUC) of Memorial Sloan Kettering Cancer Center. Subcutaneous tumorswere established in intact male mice through injection of LNCaP-AR orVCaP human prostate cancer cell lines. LNCaP-AR cells (2×10⁶) in 100 μLof 1:1 media/Matrigel (BD Biosciences, San Jose, Calif.) weresubcutaneously injected into the hindlimb area of 6-8 week old, male,athymic nude mice (NCI, Fredrick, Md.) or SCID mice(C.B-Igh-l^(b)/IcrTac-Prkdc^(scid), Taconic, Hudson, N.Y.). Also VCaPcells (2×10⁶) were injected into SCID mice (Taconic). Tumor growth wasmonitored by caliper measurement weekly. When the volume of tumorsreached approximately 100 mm³, the animals were randomly assigned todifferent cohorts for further experiments.

(iii) Treatments:

VTP: An anesthetic cocktail of 150 mg/kg ketamine and 10 mg/kg xylazinewas administered intraperitoneally prior to treatment and wassupplemented with inhaled isoflurane. A single dose of carprofen (5mg/kg) and 1 mL of subcutaneous warm saline were administered. WST11 wasreconstituted in sterile 5% dextran in water at 2 mg/mL under lightprotected condition and the aliquots were stored at −20° C. At the dayof VTP treatment, an aliquot was thawed and filtered through 0.2 μm discsyringe filter (Sartorius Stedin Biotech North America, Bohemia, N.Y.).The mice were intravenously infused with WST11 via tail vein (9 mg/kg)followed immediately by 10 minutes laser (Ceramoptec, Bonn, Germany)illumination (755 nm, 100 mW/cm for transcriptome analyses and 150 mW/cmfor in vivo studies) through a 1 mm frontal fiber (MedLight S.A.,Ecublens, Switzerland). The light field was arranged to cover the entiretumor area plus 1 mm rim using red-light aiming beam.

Androgen blockade therapy (ADT): Single dose of degarelix wasadministered at 0.5 mg per mouse at 3 days before VTP treatment viasubcutaneous or intraperitoneal injection. Drug administration wasinitiated when tumor size reached ˜100 mm³.

(iv) PSA detection in serum: Free PSA (inactive PSA) and total PSA(active+inactive PSA) were measured with a dual-label immunofluorometricassay (DELFIA Prostatus™ PSA Free/Total PSA; Perkin-Elmer Life Sciences)according to the manufacturer's recommendations. This assay measuresfree PSA and complexed PSA in an equimolar fashion [Ulmert et al., 2012;Mitrunen et al., 1995], and the cross-reactivity of PSA-ACT for free PSAis less than 0.2% [Pettersson et al., 1995]. The lower limits ofdetection are 0.1 ng/mL for both total PSA and free PSA. For detection,the 1235 automatic immunoassay system from Perkin-Elmer Life Sciences(Waltham, Mass.) was used.

(v) Histology and immunohistochemistry: All tumor specimens were fixedin 10% buffered formalin (Fisher Scientific, Pittsburgh, Pa.), processedroutinely, embedded in paraffin, sectioned at 5-micron thickness, andstained with hematoxylin-eosin (H&E). Immunohistochemistry (IHC) oftumors was performed on 5 micron formalin-fixed paraffin embedded (FFPE)section following heat induced epitope retrieval (HIER) in a buffer atpH 9.0. AR staining with anti-AR antibody (at 0.66 μg/ml, Abcam,Cambridge, Mass.) and TUNEL staining for cell death with terminaldeoxynucleotidyl transferase dUTP nick-end labeling (Roche Diagnostics,Indianapolis, Ind.) was performed using Discovery XT processor (VentanaMedical Systems, Inc., Tucson, Ariz.) at the Molecular Cytology corefacility. IHC staining for CD31 and Ki67 markers was performed on FFPEsections at the Laboratory of Comparative Pathology on a Leica Bond RXautomated stainer (Leica Biosystems, Buffalo Grove, Ill.). FollowingHIER at pH 9.0, the primary antibody against CD31 (DIA-310, Dianova,Hamburg, Germany) or Ki67 (ab16667, Abcam, Cambridge, Mass.) was appliedat a concentration of 1:250 and 1:100, respectively, followed byapplication of a polymer detection system (DS9800, Novocastra BondPolymer Refine Detection, Leica Biosystems). For all IHC stains andTUNEL, the chromogen was 3,3-diaminobenzidine tetrachloride (DAB), andsections were counterstained with haematoxylin. For quantification ofCD31, Ki67 and TUNEL staining, whole slide digital images were generatedon a scanner (Pannoramic 250 Flash III, 3DHistech, 20×/0.8NA objective,Budapest, Hungary) at a resolution of 0.2431 μm per pixel. Stainingquantification was performed with QuPath 0.1.2 software (Centre forCancer Research & Cell Biology, Queen's University Belfast, UK). ForCD31 and Ki67, the region of interest (ROI) was defined as viable tumortissue excluding necrosis. For TUNEL, the ROI was defined as total tumortissue including necrosis. For CD31 and TUNEL, the positive area,defined as the ratio of DAB stained pixels to total ROI area, wasmeasured using the positive pixel count algorithm. For Ki67, the ratio(percentage) of cells with positive nuclear staining to total cellnumber was measured with the positive cell detection algorithm. ROIselection, algorithm optimization and validation, and qualitativeexamination of H&E slides, were performed by a board-certifiedveterinary pathologist (SM).

(vi) GSEA for transcriptome analysis of LNCaP-AR xenografts followingVTP treatment: LNCaP/AR xenografts were established in intact SCID miceby injecting 2 million cells as described previously [Chen et al., 2004]and, once established, were treated with VTP at 9 mg/kg WST11 followedby 753 nm illumination at 100 mW/cm² laser fluence. Tumors werecollected at 3, 6, or 24 hours, 1 week and 8.5 weeks post VTP and RNAwas isolated following the standard protocol using TRIzol (FisherScientific). Expression profiling was performed using Illumina HT-12Expression BeadChip array and the data was analyzed using PartekGenomics Suite (Partek Inc., St. Louis, Mo.). The microarray data thenunderwent secondary analysis by GSEA [Subramanian et al., 2005] usinggene sets from the Hallmark and C2, Canonical Pathways collections(Molecular Signature Databases v6.0 (MSigDB); Broad Institute at httpsite software.broadinstitute.org/gsea/msigdb). GSEA| MSigDB. Accessed 19Jun. 2017. Microarray data has been deposited in the National Center forBiotechnology Information Gene Expression Omnibus (GEO) (GSE109681).

(vii) Statistical analysis: Two-way ANOVA test using GraphPad Prism(GraphPad Software, La Jolla, Calif.) was used for therapeutic efficacyin affecting tumor growth, and One-way ANOVA for PSA and a Mann-Whitneytest for CD31, Ki67 or TUNEL staining quantification. Differences with pvalues<0.05 were considered statistically significant.

EXAMPLES Example 1. Transcriptome Analysis of WST11 VTP-Treated Tumorsby GSEA Revealed an Enrichment of Androgen Response Pathways

To identify potential druggable pathways active in PCa that could beexploited for combination therapy with VTP, we analyzed thetranscriptome of LNCaP-AR xenograft tumors following acute response toVTP exposure. Using the LNCaP-AR prostate cancer model system, weemployed microarrays and Gene Set Enrichment Analysis (GSEA) to identifypotential druggable pathways active in tumors exposed to VTP. Three tosix hours post-VTP, androgen responsive gene sets were enriched,suggesting that the androgen receptor (AR) may be a viable target incombination with VTP. Unbiased GSEA identified statistically significantenrichments with gene sets related to hypoxia, HIF1A, and VEGFR pathwaysat three to six hours post-VTP treatment (FIGS. 1A-1B), effects thathave previously been shown to be associated with photodynamic therapies(PDT) [Broekgaarden et al., 2015]. Interestingly, AR signaling gene setswere also up-regulated in VTP-treated tumors compared to control mice,suggesting that the AR may be a viable target for combination therapywith VTP.

Example 2. ADT and VTP Displays Potential Enhanced Effects: LNCaP-ARModel

We tested this hypothesis in mice bearing LNCaP-AR xenograft tumors byusing the AR pathway inhibitors degarelix (to achieve pharmacologicalcastration) or enzalutamide (AR antagonist), alone or in combinationwith VTP. Degarelix was administered as a single 0.5-1 mg dose 3 daysprior to initiation of VTP, while enzalutamide (30 mg/kg) was givendaily for two weeks total both before and after VTP (FIG. 2, Panel A).Nude mice bearing LNCaP-AR tumors were randomly assigned to 6 cohorts:control (n=7), degarelix (n=9), enzalutamide (n=10), VTP (n=8),degarelix+VTP (n=9), and enzalutamide+VTP (n=9) and tumor size wasmeasured weekly. The results in FIG. 2, Panel B show that compared toeither AR pathway inhibitor or VTP used alone, degarelix or enzalutamidein combination with VTP significantly inhibited tumor growth.

Example 3. Combination Therapy of ADT with WST11NTP Suppressed TumorGrowth to a Greater Extent than Either Treatment Alone in a LNCaP-ARHuman Prostate Cancer Model in Nude Mice and SCID Mice

To test our hypothesis that the preemptive blocking of androgensignaling upregulation induced by VTP might improve the outcome of tumorgrowth control, we tested combination of VTP with an androgen signalingpathway inhibitor in widespread clinical use for the treatment of PCa.To establish preexisting AR inhibition, treatment with degarelix wasinitiated three days prior to administering VTP to PCa xenograft tumors.Prior studies with WST11 VTP established that components of the immuneresponse contributed to the anticancer activity of VTP [Preise et al.,2009]. Therefore, the efficacy of the combination therapy was comparedagainst LNCaP-AR tumors in both athymic nude (T cell deficient) (FIG.3A) and severe combined immunodeficiency (SCID) (both T and B celldeficient) (FIG. 3B) mice. Tumor-bearing nude mice were randomlyassigned to four cohorts: control, degarelix, VTP, and degarelix and VTPcombination. The combination of degarelix and VTP resulted instatistically significant improved tumor growth control compared toeither degarelix (p<0.01) or VTP alone (p<0.005) (FIG. 3A). As in thenude mouse, the combination of degarelix and VTP led to superior controlof LNCaP-AR tumor growth in SCID mice (p<0.0001 for either monotherapyvs. combination) (FIG. 3B).

Example 4. Combination Therapy of ADT with WST11NTP Delayed Tumor Growthto a Greater Extent than Either Treatment Alone in the VCaP-AR HumanProstate Cancer Model in SCID Mice

SCID mice bearing VCaP tumors were randomly assigned to 4 cohorts:control (n=7), degarelix (n=9), VTP (n=8), degarelix+VTP (n=9), andtumor size was measured weekly. One dose of degarelix was given 3 daysprior to VTP treatment. The combination of degarelix and VTP wassignificantly more effective than VTP alone (p<0.005) or degarelix alone(p<0.0001) (FIG. 4) in delaying the growth of VCaP, a human PCa modelwith AR gene amplification that also expresses the constitutively activeAR splice variant AR-V7.

Example 5. Combination Therapy of ADT and VTP was More Effective thanVTP Alone in Downregulation of Total PSA Levels and Induction ofApoptosis/Necrosis

To verify that AR activity was inhibited by the treatments, the levelsof total PSA (tPSA) in serum of mice bearing LNCaP-AR tumors weremeasured. Serum tPSA values were determined in separate cohorts of miceat one, three, or seven days post-WST11 VTP (four, six or ten dayspost-degarelix). As shown in FIG. 5A, fold changes of tPSA valuesdeclined by either VTP or degarelix alone, but the sharpest drop in tPSAlevels was seen with the combination of degarelix and WST11 VTP (p<0.05vs. control across all time points).

In parallel, the histology of control, VTP, degarelix and degarelix+VTPcombination treated tumors on days three and seven post-VTP was assessedby both hematoxylin/eosin (HE) and TUNEL assay to detect cell death(apoptotic and/or necrotic cells). As shown in FIG. 5B, VTP-treatedtumors displayed partial cell death characterized by large foci of TUNELstaining, but with significant TUNEL negative areas. Tumors treated withcombination therapy appeared to display more extensive areas of TUNELstaining. Although not statistically significant compared to VTP alone,there were fewer tumor cells that escaped death in the combinationgroup, suggesting that increased cell death underlies the effect ontumor inhibition. In contrast, degarelix alone treated tumors exhibitedlittle TUNEL staining, but still showed reduced Ki67 signal compared tocontrols, as expected (p<0.05, FIG. 5C). Nuclear AR staining wasinversely correlated with TUNEL, suggesting that viable AR-positivecells had escaped focal therapy effects of WST11 VTP alone. Notably, thetumors treated with combination therapy were absent of AR staining,suggesting that remaining viable tumor cells we few in number.

Example 6. ADT Reduces the Number of Vessels in Tumors

CD31 is primarily a marker for endothelial cells which can help evaluatethe degree of intratumoral vessel formation. This is achieved by CD31H&E and immunohistochemical (IHC) staining of control anddegarelix-treated tumors. As shown in FIG. 6A, the image analysisshowing positive pixels in red (lower panels) indicates thatdegarelix-treated tumors (right panel) appear to have fewer vessels thantumors in the control group (left panel). The quantification of CD31positive areas in the control group (n=12) and the degarelix-treatedgroup (n=11) in FIG. 6B show that degarelix treatment resulted in a 38%decrease in CD31 staining area compared to controls (p<0.05).

DISCUSSION

The effective adoption of prostate cancer screening has led to earlierdetection of small, clinically significant prostate cancers amenable tothe newly developed treatment strategies for partial gland ablationwhich are well tolerated and associated with fewer adverse side effectsthan aggressive radical therapies such as surgery and radiation.

Positive oncologic outcomes in clinical studies of VTP has led to therecent approval of TOOKAD® Soluble (WST11 or padeliporfin) for thetreatment of low-risk PCa and highlights the potential of VTP to serveas an alternative to active surveillance or radical therapies (Azzouziet al., 2015; Azzouzi et al., 2017; Lebdai et al., 2017). To potentiallyextend VTP treatment to larger cohorts of patients, VTP clinical trialsare planned for patients with localized PCa of intermediate risk, GradeGroup 2 [Gleason score 7 (3+4)].

REFERENCES

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1. A method for treatment of prostate cancer by combination therapycomprising administering to a patient in need thereof: (i) atherapeutically effective amount of an androgen-deprivation therapy(ADT) agent (hereinafter “ADT agent”); and (ii) a therapeuticallyeffective amount of a bacteriochlorophyll derivative (Bchl-D) followedby vascular-targeted photodynamic therapy (VTP) (hereinafter “Bchl-DVTP”), wherein said ADT agent is enzalutamide that is administered onceor in repeated doses, during several days, before and optionally afteradministration of said Bchl-D. 2-6. (canceled)
 7. The method of claim 1,wherein the Bchl-D is an anionic bacteriochlorophyll derivativeoptionally conjugated with an RGD-containing peptide orRGD-peptidomimetic residue.
 8. The method of claim 7, wherein the Bchl-Dhas the formula I:

wherein M represents 2H or Pd; R₁ is O—R₄ or —NHR₅, wherein R₄ isselected from the group consisting of H, H⁺, an ammonium group or amonovalent metal cation, and R₅ is an RGD-containing peptide or RGDpeptidomimetic residue; R₂ is —O—C₁-C₆ alkyl; R₃ is —NH—(CH₂)_(n)—SO₃⁻R₆ ⁺, wherein n is 2 or 3, and R₆ ⁺ is a monovalent metal cation; orpharmaceutically acceptable salts or optical isomers thereof.
 9. Themethod of claim 8, wherein the monovalent metal cation represented by R₄and R₆ ⁺ each or both are Na⁺ or K⁺, and R₂ is methoxy.
 10. The methodof claim 9, wherein R₁ is O—R₄, R₂ is methoxy, R₃ is —NH—(CH₂)_(n)—SO₃⁻R₆ ⁺, wherein n is 2, and R₄ and R₆ ⁺ are K⁺.
 11. The method of claim10, wherein the Bchl-D of formula I is the compound Palladium3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13¹-(2-sulfoethyl)amide dipotassium salt (herein designated WST11) or the compound3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13¹-(2-sulfoethyl)amide dipotassium salt (herein designated STL-7012).
 12. The method ofclaim 11, wherein the area to be treated by said VTP is locallyilluminated immediately after the administration of the Bchl-D.
 13. Themethod of claim 11, wherein the area to be treated by said VTP islocally illuminated at a time of up to 30 min after the administrationof the Bchl-D.
 14. The method of claim 9, wherein in the Bchl-D offormula I R₁ is NH—R₅, and R₅ is a cyclic RGD-containing peptide or acyclic RGD-peptidomimetic residue.
 15. The method of claim 14, whereinthe Bchl-D is STL-6014. 16-17. (canceled)
 18. The method of claim 1,wherein the Bchl-D derivative is WST11.
 19. The method of claim 18,wherein the WST11 is administered once and enzalutamide is administeredonce before and one or several times after the administration of WST11at determined time intervals. 20-21. (canceled)
 22. The method of claim1, wherein the prostate cancer is a low risk prostate cancer.
 23. Themethod of claim 1, wherein the prostate cancer is an intermediate riskprostate cancer.
 24. The method of claim 1, wherein the prostate canceris a high risk prostate cancer.
 25. The method of claim 1, wherein theprostate cancer is castration-sensitive.
 26. The method of claim 1,wherein enzalutamide is administered as a single dose several daysbefore administration of said Bchl-D, which is followed by VTP.
 27. Themethod of claim 1, wherein enzalutamide is administered in repeateddoses, during several days, before and after administration of saidBchl-D, which is followed by VTP.
 28. The method of claim 18, whereinenzalutamide is administered daily for two weeks total both before andafter VTP, starting several days before administration of WST11.
 29. Themethod of claim 28, wherein administration of enzalutamide starts 3 daysbefore administration of WST11, which is followed by immediate laserlocal illumination of the area at 755 nm.